Infectious bursal disease (IBD) is an acute contagious viral disease of young chickens often known as Gumboro disease (Kibenge et al., J Gen Virol. 69(Pt 8):1757-1775, 1988; Lasher et al., Avian Dis. 41(1):11-19, 1997). The etiological agent, IBD virus (IBDV), has a predilection for the cells of the bursa of Fabricius where the virus infects actively dividing and differentiating lymphocytes of the B-cell lineage (Burkhardt et al., Arch Virol. 94(3-4):297-303, 1987). Thus, IBD is a fatal immunosuppressive disease causing heavy losses to the poultry industry (Eterradossi et al., Arch Virol. 143(8):1627-1636, 1998).
The first outbreak of IBDV was reported in commercial chicken flocks in Delaware, USA (Cosgrove, Avian Dis. 6:385-389, 1962). The IBDV strains, which were isolated during the outbreak, now referred to as classical serotype I isolates. The disease was also first report in Europe in 1962 (Faragher, Vet. Bull. 42:361-369, 1972). And from 1966 to 1974, IBD was reported in the Middle East, Southern and Western Africa, India, the Far East and Australia (Faragher, 1972; Firth, Aust Vet J. 50(3):128-130, 1974; Jones, N Z Vet J. 34(3):36, 1986; van den Berg, Avian Pathol. 29:175-194, 2000). In most cases, the IBDV strains that associated with the outbreaks were of low virulence and caused only 1 to 2% of specific mortality (van den Berg, 2000).
However, a new IBDV strain (antigenic variant) emerged and able to cause up to 5% specific mortality in USA (Rosenberger and Cloud, Avian Dis. 33(4):753-759, 1989). The antigenic variant was recovered from flocks with selection pressure of field vaccination against classical IBDV serotype I (Snyder, 1990). Although being antigenic variant these isolates have only minor amino acid changes and do not form a separate serotype.
Nevertheless, these changes occur at the VP2 conformation-dependent antigenic epitopes that are responsible for stimulating virus neutralizing antibodies (Bayliss et al., J Gen Virol. 71(Pt 6):1303-1312, 1990). Currently, variant form of IBD has been reported outside Central America particularly in countries such as China (Cao et al., Avian Dis. 42(2):340-351, 1998), South America (Banda et al., Avian Dis. 47(1):87-95, 2003) and Australia (Sapats and Ignjatovic, Arch Virol. 145(4):773-785, 2000).
Since variant IBDV causes only changes at the bursa and depending on the immune status of the chickens, the disease is often manifested with subclinical signs, it is difficult to detect variant IBDV in commercial flocks. Hence, variant IBDV may be common in many countries in the world but remains undiagnosed. A second serotype—serotype II of IBDV was identified in 1987 (McNulty and Saif, Avian Dis. 132(2):374-375, 1988). Serotype II IBDV isolates are apathogenic and are recovered mainly from turkeys (Ismail et al., Avian Dis. 32(4):757-759, 1988).
In the 1990s, IBDV isolates, which were able to break through levels of maternal antibodies that normally were protective, were reported in Europe (Chettle et al., Vet Rec. 125(10):271-272, 1989). These isolates, the so called very virulent IBDV are causing more severe clinical signs during an outbreak which mortality approaching 100% in susceptible flocks, and are now found almost world-wide (van den Berg, 2000). The emergence of very virulent strains of IBDV has complicated the immunization programs against the disease.
Early vaccination may result in failure due to interference with the maternal antibody, whilst its delay may cause field virus infections. Currently, outbreaks of vvIBDV have been reported throughout various countries in the world (Banda et al., Avian Dis. 47(1):87-95, 2003; Cao et al., Avian Dis. 42(2):340-351, 1998; Chai et al., Arch Virol. 146(8):1571-1580, 2001; Chettle et al., 1989; Eterradossi et al., Zentralbl Veterinarmed B. 39(9):683-691, 1992; Hoque et al., J Biochem Mol Biol Biophys. 6(2):93-99, 2002; Liu et al., Virus Genes. 24(2):135-147, 2002; Majo et al., Avian Dis. 46(4):859-868, 2002; Rudd et al., Aust Vet J. 81(3):162-164, 2003; Scherbakova et al., 1998; Ture, et al., Avian Dis. 42(3):470-479, 1998; Zorman-Rojs et al., Avian Dis. 47(1):186-192, 2003).
In designing an effective disease control program one should consider the diagnostic methods use to diagnose disease caused by infectious agent. Currently, IBD can be diagnosed based on virus isolation, electron microscopy, immunofluorescence, virus neutralization, monoclonal antibody assays, and/or enzyme-linked immunosorbent assay (Jackwood et al., Clin Diagn Lab Immunol. 3(4):456-463, 1996; Jackwood et al., Avian Dis. 40(2):457-460, 1996; Liu et al., J Virol Methods. 48(2-3):281-291, 1994; Lukert and Saif, Infectious bursal disease. In: Diseases of Poultry, 10th edn (Eds. Calnek et al.), Iowa State University Press, Ames, Iowa, pp. 721-738, 1997; Wu et al., Avian Dis. 36(2):221-226, 1992). However, these methods have one or more disadvantages such as time consuming, labour intensive, expensive and of low sensitivity (Wu et al., 1992).
Recently, the reverse transcriptase polymerase chain (RT-PCR) has been used to detect IBDV based on the amplification of the central hypervariable region of the VP2 region (Tham et al., J Virol Methods. 53(2-3):201-212, 1995; Jackwood and Nielsen, Avian Dis. 41(1):137-143, 1997). Subsequently, RT-PCR assay followed by restriction fragment length polymorphism (RFLP) also has been used to detect and differentiate IBDV strains (Jackwood and Sommer, Avian Dis. 41(3):627-637, 1997; Jackwood and Sommer, Avian Dis. 43(2):310-314, 1999; Hoque et al., Avian Pathol. 30:369-380, 2001; Ture et al., Avian Dis. 42(3):470-479, 1998; Zierenberg et al., 2001).
Although, this method able to differentiate different IBDV strains, it is not automated and time consuming. Both radioactive and non-radioactive based nucleic acid probes that can differentiate IBDV strains have been used in the detection of IBDV (Akin et al., Vet Diagn Invest. 5(2):166-173, 1993; Davis and Boyle, Avian Dis. 34(2):329-335, 1990). However, apart for academic interest, their use in diagnosing IBD is uncommon.
Fluorescence-based real-time PCR assays have been developed to provide a rapid and sensitive method for quantifying nucleic acids (Gibson et al., Genome Res. 6(10):995-1001, 1996; Heid et al., Genome Res. 6(10):986-994, 1996; Desjardin et al., J Clin Microbiol. 36(7):1964-1968, 1998). In this assay, reactions are monitored by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. There are currently 2 general approaches in real-time PCR depending on the types of fluorescence dyes. The simplest method uses fluorescent dye, SYBR Green I that bind specifically to double stranded DNA (Morrison et al., Biotechniques. 24(6):954-958, 960, 962, 1998). The major problem with SYBR Green I-based detection is that non-specific amplifications cannot be distinguished from specific amplifications. However, specific amplification can be verified by melting curve analysis (Ririe et al., Anal Biochem. 245(2):154-160, 1997).
The other dyes (TaqMan, Molecular Beacons, Scorpion) rely on the hybridization of fluorescence labeled probes to the correct amplicon (Wittwer et al., Biotechniques. 22(1):130-131, 134-138, 1997; Bonnet et al., 1999). Accumulation of PCR products is detected by monitoring the increase in fluorescence of the reporter dye. The threshold cycle (Ct) is defined as the fractional cycle number at which the reporter fluorescence generated by the accumulating amplicons passes a fixed threshold above baseline (Mackay et al., Nucleic Acids Res. 30(6):1292-1305, 2002).
Hence, a plot of the log of initial target copy number for a set of standards versus Ct is a straight line whereby the higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed (Higuchi et al., Biotechnology (NY). 11(9):1026-1030, 1993; Gibson et al., 1996; Heid et al., 1996; Desjardin et al., 1998). It has also been established that primers combination playing an important role for the Ct value prediction, where the approach is similar to the analysis of single-nucleotide polymorphism (Frederique et al., 2003; Christy et al., 2002; Srinivas et al., 2000). Thus, several recent studies have used SYBR Green I based real-time PCR to differentiate different serotypes or strains of organisms based on Ct and/or Tm values (Aldea et al., J Clin Microbiol. 40(3):1060-1062, 2002; Beuret, J Virol Methods. 115(1):1-8, 2004; Nicolas et al., J Microbiol Methods. 51(3):295-299, 2002; Shu et al., J Clin Microbiol. 41(6):2408-2416, 2003). A quantitative real-time PCR assay based on TaqMan has been developed to detect IBDV (Moody et al., J Virol Methods. 2000 85(1-2):55-64, 2000). In other recent studies by Jackwood and Sommer (Virology. 304(1):105-113, 2002) and Jackwood et al. (Avian Dis. 47(3):738-744, 2003), TaqMan based real-time PCR was shown to be able to detect vaccine and wild type IBDV strains in infected chickens. However, the assay is expensive and more complex compared to the method established in this study. In addition, the application of the method to differentiate very virulent and vaccine strains IBDV is not known.